Adoptive T Cell Therapy: - PDF Free Download

Adoptive T Cell Therapy: A Paradigm for Combination Strategies Cassian Yee MD Professor Melanoma Medical Oncology Immunology Director Solid Tumor Cell Therapy cyee@mdanderson.org skype name: tcelltherapy

Adoptive T Cell Therapy: Green Eggs and Ham Cassian Yee MD Professor Melanoma Medical Oncology Immunology Director Solid Tumor Cell Therapy cyee@mdanderson.org skype name: tcelltherapy

Immunotherapy Green Eggs and Ham

Adoptive T Cell Therapy Immune Checkpoint Blockade

Adoptive T Cell Therapy: Beer-Margarita Paradigm Cassian Yee MD Professor Melanoma Medical Oncology Immunology Director Solid Tumor Cell Therapy cyee@mdanderson.org skype name: tcelltherapy

Two great tastes that taste great together! The Beer Beer Margarita paradigm The Margarita

How to make a beer? TIL Tumor-infiltrating Lymphocytes TIL enrichment IL-2 TCR/CAR Engineered T cells Chimeric TCR + zeta Chimeric Ig + zeta Receptor Transfer ETC Endogenous T cell Therapy Cloning Cell sorting Antigen-specific T Cell Enrichment

TIL Tumor-infiltrating Lymphocytes TCR/CAR Engineered T cells No knowledge of tumor antigen required Effective for melanoma Potentially other TIL+ tumors Effector cell enrichment Serious toxicities, Selection bias Potential off-the-shelf Genetic enhancement possible Leukemia Potentially other malignancies Serious toxicities Regulatory/ Safety

Endogenous T Cell Therapy (ETC) Source Peripheral Blood < 0.001 % TCR repertoire self-selected affinity Unbiased from TIL Accessible Peripheral blood Low morbidity/outpatient Regulatory simplicity Genetic modification Rapid deployment Discovery Implementation Flexibility Time and labor-intensive technically challenging

Endogenous T Cell therapy ETC Source Enrichment Expansion Peripheral Blood APC + peptide STIM 1 < 0.001 % > 80 % > 80 % 10 6 10 10

CD8+ T cell clones HD Cytoxan LD IL-2 CD8+ T cell TCR

Lymphodepletion building a better environment CD8+ T cell clones HD Cytoxan LD IL-2 PNAS 2012

Efficacy

Efficacy Patient Target Toxicity Disease Sites Response 2140-1 Tyrosinase F,N,R Cervical,supraclavicular LN, Chest Wall, Breast Pulmonary nodules 2140-2 Tyrosinase F Mediastinal, Pulmonary nodules PD MR 2140-3 MART-1 F,N,R Mesenteric LN, scapular subcutaneous dz 2140-4 MART-1 F, N, R Pulmonary, inguinal, subcutaneous 2140-5 MART-1 F, N,R Right and left kidneys, adrenal, liver 2140-6 MART-1 F, N, R Mediastinal, supra clavicular, mammary chain, periportal, portacaval nodes. CR (> 12 mths) PR- PR- PNAS 2012

CTL frequency/10^6 Persistence Complete Response Effector Memory 250000 200000 150000 100000 50000 0 9.4% 5.3% Central Memory 1.1% CD45 RO+ CD28++ CD127-hi 0 D18 50 100 150 200 250 300 Days post Infusion Progressive Disease 5000 4000 3000 2000 1000 Effector Memory CD45 RO+ CD28- CD127-lo 0 0 50 100 150 200 250 300 Days post Infusion

All T cells are equal, but some are more equal than others Apologies to G. Orwell 18

A more equal T cell CD8+ T cell TCR CD127 CD28

Can we prospectively isolate central memory CD8 T cells? Naïve Central Memory Effector Memory Terminal Effector cytolytic proliferative CD28+ CD127+ CD45RA+ CD28++ CD127+ CD45RO+ CD28 - CD127- CD45RO+ CD28 - CD127- CD45RA+

Cytokine Modulation Enriches for Central Memory T cells Peripheral Blood Stim # 1 APC + peptide Frequency Surface Phenotype Functional Assays Affinity γ- chain receptor cytokines IL-2 IL-7 IL-15 IL-21 J Immunol 2005 Blood 2008 Science Transl Med 2013

Interleukin-2 (pg/ml) % of Max FL2-H: PE-M26-tet FL2-H: PE-M26-tet 10 4 IL-2, -7 or -15 10 4 IL-21 10 3 0.340.34 10 3 1919.0 10 2 10 2 10 1 10 1 10 0 10 0 10 1 10 2 10 3 10 4 FL1-H: FITC 10 0 10 0 10 1 10 2 10 3 10 4 FL1-H: FITC 100 80 60 40 20 0 1 10 100 1000 10000 FL4-H: APC-CD28 CD28 400 350 300 250 200 T2 unpulsed T2 + M27pept T2 + M27pept +CTLA4-Ig Longer-Lasting (IL-2 producing, helperindependent CTL) 150 100 50 0 IL-2, -7, -15 IL-21 treated 2005 J Immunol 2008 Blood 2013 Sci Trans Med

CD8+ T cell TCR CD127 CD28 Naïve Central Memory Effector Memory Terminal Effector cytolytic proliferative CD28+ CD127+ CD45RA+ CD28++ CD127+ CD45RO+ CD28 - CD127- CD45RO+ CD28 - CD127- CD45RA+

Priming with IL-21 Generates Central Memory-type T cell IL-2 CD8+ T cell TNF-a TCR IFN-g CD28++ CD127+ CD45RO+ CD127 CD28 helper-independent CTL (Tcm) J Immunol 2005 Blood 2008 Science Transl Med 2013

IL-2 TNF-a Intrinsic (beer) CD127 CD8+ T cell IFN-g TCR CD28 Extrinsic (margarita) Pre- Chemotherapy XRT Select Post- Cytokine help - Low-dose IL-2 - High-dose IL-2 Other g-chain receptor cytokines Anti-CTLA4, Anti PD-1 Agonist antibodies Vaccine + adoptive therapy Oncolytic virotehrapy

Blockade of Immune Checkpoint Inhibitor CTLA4

Blockade of Immune Checkpoint Inhibitor CTLA4 Transferred tumor antigen-specific T cells enhanced proliferative potential Endogenous tumor antigen-specific T cells lower threshold for activation Leads to antigen-spreading Multivalent response Eradicate / Modulate function CTLA4+ Tregs

Phase I/II Trial of Adoptive T Cell Therapy in Combination with Immune Checkpoint Blockade IL-2 TNF-a CD127 CD8+ T cell IFN-g TCR CD28 Metastatic Melanoma HLA-A2+ MART-1 10 10 cells/m 2 Aude Chapuis IL-21-CD8+ T cells Anti-CTLA4 (D1) Anti-CTLA4 (D22/W3) Anti-CTLA4 (D43/W6) Anti-CTLA4 (D64/W9) LD IL-2 Chapuis, Greenberg

Clinical Responses In Patients with Metastatic Melaonma Receiving Adoptive T Cell Therapy And Concurrent Anti-CTLA4 Patient Prior Ipilimumab treatment failure 1 YES 2 YES 3 NO 4 YES 5 NO 6 NO 7 NO 8 NO 9 NO 6 weeks (-2) (-2) (0) PD (69) PD (+30) PD (+31) (-45) (+8) (-41) 12 weeks (-34) (-6) (+19) (+17) PR (-90) (+21) 16/19 weeks (-7) PD (+27) PR (-71) 28 weeks PR (-80) PD (+25) CR (-100) 40 weeks PR (-90) 84 weeks CR (-100) 10 NO PR (-76) PR (-79)

Clinical Responses In Patients Receiving Adoptive T Cell Therapy And Concurrent Anti-CTLA4 Patient Prior Ipilimumab treatment failure 1 YES 2 YES 3 NO 4 YES 5 NO 6 NO 7 NO 8 NO 9 NO 6 weeks (-2) (-2) (0) PD (69) PD (+30) PD (+31) (-45) (+8) (-41) 12 weeks (-34) (-6) (+19) (+17) PR (-90) (+21) 16/19 weeks (-7) PD (+27) PR (-71) 28 weeks PR (-80) PD (+25) CR (-100) 40 weeks PR (-90) 84 weeks CR (-100) 10 NO PR (-76) PR (-79) WHO RECIST Criteria

Clinical Response Adoptive CTL therapy + anti-ctla4

Persistence

Acquisition of Central Memory Markers Correlates with Clinical Response

Spots/100,000 PBMC Antigen-spreading PATIENT #1 50% clinical response at 24 weeks 25 Mart1 20 15 10 5 0 7 9 11 9 11 13 2 4 MART-1 6 8 10 NY-ESO-1 12 13 15 17 gp100 19 21 23 2 4 6 Tyrosinase 8 Mage A3 INF D+189 INF D+83 INF D+43 INF D-6 INF #2 D+50 INF #2 D+29 INF #2 D+0 INF #1 D+0 Ilana Roberts

Responders Non- Responders

Conclusions/Future Directions Combination of T cell therapy and anti-ctla4 leads to establishment of long-lived central memory T-cells. Evidence of epitope spreading was observed in patients with tumor regression/stable disease. Established a highly effective outpatient strategy > 60% disease control in patients with metastatic dz Phase II study -> 30 patients (MD Anderson Cancer Center)

Microbrewery or Beer Factory 43

T Cell Therapy Source Enrichment Expansion Peripheral Blood APC + peptide STIM 1 < 0.001 % > 80 % > 80 % 10 6 10 10 12-14 weeks

FITC-A <FITC-A> Clinical Grade pmhc-multimer-based Sorting Specimen_001_E2_E02.fcs FSC-A, SSC-A subset 10 5 22.6 0.37 cd4 isolation exp3_2537-so2 EB REP INF1.fcs FSC-H, SSC-A su 0.37% 2.37 93.6% 10 5 10 4 10 4 10 3 10 3 10 2 0 77 8.34e-3 0 10 2 10 3 10 4 10 5 APC-A 10 2 0 3.58 0.45 0 10 2 10 3 10 4 10 5 <APC-A>

T Cell Therapy Source Enrichment Expansion Peripheral Blood APC + peptide STIM 1 < 0.001 % > 80 % > 80 % 10 6 10 10 4 weeks

T Cell Therapy: Enabling Technologies Turnkey Operation Source Selection Expansion Peripheral Blood APC + peptide STIM 1 +IL-21 MART-1 NY-ESO-1 MAGE-A3 MAGE-A1 PRAME WT-1 Melanoma Lung Head and Neck Hepatocellular GI/ Gastric Survivin

Beyond Melanoma Anti-CTLA4 Anti-PD1 Hodi et al, NEJM 2010 Topalian S L et al. JCO 2014 Lawrence et al, Nature. 2013

A NY-ESO-1 is expressed in MRCL with high prevalence and homogeneity B C Pollack et al Cancer, 2011 PLOS One 2012

% Tetramer Tetramer Tetramer Tetramer NY-ESO-1+ T-cells targeting HLA A*2402/NY-ESO-1 I (Metastatic Breast Cancer) 0.94% 0.92% 3.55% 4 3.5 3 CD8 CD8 CD8 2.5 2 1.5 1 0.5 0 Anti-CTLA4-3 3 9 15 21 27 33 39 45 51 57 63 HD-Cytoxan (Day -3) Infusion #1 (Day 0) LD- Cytoxan (Day 31) Infusion #2 (Day 34) A. Ribas J Glaspy C Yee

Greg Lizee Identifying new antigens

Generation of SLC45A2 specific CTLs Schematic steps DC stimulation of CD8 T cells SLA45A2 peptide (R/S) 1 st stimulation 2 nd stimulation Tetramer staining Sorting of tetramer+ CD8 T cells REP Cloning Cloning Tetramer staining CRA CRA CRA REP REP REP SLC45A2 specific CD8 T cells Jungsun Park

Generation of SLC45A2 specific CTLs Tetramer staining 2.25% 2.31% 6.21%

2381 2508 2400 2412 2382 2559 2461 2333 Mel 888+A2 Specific lysis (%) Specific lysis (%) Specific lysis (%) Specific lysis (%) Specific lysis (%) Specific lysis (%) Specific lysis (%) Specific lysis (%) Generation of SLC45A2 specific CTLs Cytotoxic activity of SLC45A2 specific CTL Mel 2381 Mel 2508 Mel 2400 Mel 2412 40 40 40 40 30 30 30 30 20 20 20 20 10 10 10 10 0 40 30 20:1 10:1 Mel E:T 2382 ratio 5:1 2.5:1 0 40 30 20:1 10:1 Mel E:T 2559 ratio 5:1 2.5:1 0 20:1 10:1 Mel E:T 2461 ratio 5:1 2.5:1 0 20:1 10:1 5:1 Mel E:T 2333 ratio SLC45A2-neg SLC45A2-neg SLC45A2-neg 40 30 40 30 2.5:1 20 20 20 20 10 10 10 10 0 20:1 10:1 5:1 2.5:1 0 20:1 10:1 5:1 2.5:1 0 20:1 10:1 5:1 2.5:1 0 20:1 10:1 5:1 2.5:1 E:T ratio E:T ratio E:T ratio E:T ratio Expression of SLC45A2 in melanoma cell lines RT-PCR SLC45A2 GAPDH SLC45A2-specific CTL can effectively kill SLC45A2- expressing melanoma cells

Mel 526 A375 Melanocyte 4C Melanocyte 3C Specific lysis (%) Specific lysis (%) SLC45A2 is a tumor-specific differentiation antigen 100 80 SLC45A2 Mel 526 100 A375 Melanocyte 3C Melanocyte 80 4C MART-1 (#160) MART-1 Mel 526 A375 Mel 526 A375 Melanocyte 3C Melanocyte 3C Melanocyte 4C Melanocyte 4C 60 60 40 40 20 20 0 40:1 20:1 10:1 E:T ratio 5:1 2.5:1 0 40:1 20:1 10:1 E:T ratio 5:1 2.5:1 SLC45A2 MART-1 Gp100 Tyrosinase GAPDH

GBM Antigen-specific T cell Generation Project in collaboration with Immatics

Antigen-specific T cell generation Leukapheresis Dendritic Cells Pulsed with antigen Pulsed with antigen CD25 Depletion Stim #1 Stim #2 Goal is to generate autologous tumor antigen-specific cytotoxic T cells from normal donor PBMCs Stim QC Bulk Well FACSAria Fusion Sort Bulk Well Sort1 REP1 REP QC (Stain, CRA) Testing a wide variety of antigens, currently focusing on GBM-specific 19 GBM antigen peptides currently tested 6 currently in Stim 1 Freeze

Tetramer - PE Staining for tetramer positive T-cells after 2 nd stimulation with example antigen Stimulations performed in 48- well plates These bulk wells are pooled and then sorted for REP (next slide) CD8 - APC

Tetramer - PE Tetramer-guided enrichment of GBM-specific CTL population Pre-REP Post-REP CD8 - APC Sort stain (bulk well pool) About 8x10 3 (CD8 + Tet + ) T cells sorted 94x10 6 expanded (12,000-fold expansion)

Conclusions Non-melanoma solid tumor malignancies can be targeted by ETC MS/exome/RNA seq analysis yield new epitopes that can elicit peptide-specific T cells Such T cells are capable of recognizing tumor cells presenting endogenous antigen Peptide epitopes identified by this approach are immunogenic and represent potential tumor rejection antigens

The Beer Beer Margarita paradigm Cytokines Chemokines The Margarita CD8 CD4 T cm T eff T fh Th 1-50 NK NKT Genetic Modification -safety -knockdown -conditional expression CTLA4 PD1/PD-L1 GITR OX40 CD40 CD137 Vaccine Therapy Oncolytic Virotherapy Radiation Therapy Targeted Therapy Chemotherapy

Transferrable Biomarker IL-2 CD8+ T cell TNF-a TCR IFN-g CD127 CD28 Post-infusion Immunomodulation Antigen-specific T cells Pre-infusion Tetramer+ T cells From PBMC Pre-Immunomodx Analysis of antigen-specific T cells Pre-vs Post-immunomodulatory therapy Tetramer+ T cells From PBMC Post-Immunomodx - In vivo frequency - In vivo persistence - In vivo trafficking - In vivo function - Differentiation/phenotype - Transcirptional signature

Aude Chapuis Seth Pollack Yongqing Li Ivy Lai Erik Farrar Junmei Wang Eric Mortenson Nicole Cecchini This Research was supported by: CPRIT Stand Up to Cancer-AACR/CRI Cancer Research Institute Burroughs Wellcome Fund Damon Runyon Sister Institutions Network Funding (GAP)